Methods for reducing interferences

ABSTRACT

The present invention relates to a method for determining an analyte in a sample suspected to comprise said analyte, comprising a) contacting with said sample at least a first and a second capture compound for said analyte, wherein said first and second capture compounds are nonidentical capture compounds, and wherein said capture compounds compete in binding to said analyte; b) contacting said capture compounds contacted with said sample with a specifier, wherein said specifier competes in binding to said capture compounds with said analyte; c) determining the amount of complexes comprising said specifier and a capture compound; and d) determining said analyte in a sample based on the result of step c). The present invention further relates to a method for improving the specificity of an indirect immunoassay for determining an analyte, comprising replacing at least 10% of a capture compound by a non-identical capture compound; wherein the capture compound replaced competes in binding to said analyte with the capture compound introduced. The present invention further relates to kits, devices, and uses related to the aforementioned methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/621,280 (published as U.S. Publication No. 2018/0128825) filed Jun.13, 2017, which is a continuation of International Application No.PCT/EP2015/080176 filed Dec. 17, 2015, which claims priority to EuropeanApplication No. 14198784.2 filed Dec. 18, 2014, the disclosures of whichare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method for determining an analyte ina sample suspected to comprise said analyte, comprising a) contactingwith said sample at least a first and a second capture compound for saidanalyte, wherein said first and second capture compounds arenon-identical capture compounds, and wherein said capture compoundscompete in binding to said analyte; b) contacting said capture compoundscontacted with said sample with a specifier, wherein said specifiercompetes in binding to said capture compounds with said analyte; c)determining the amount of complexes comprising said specifier and acapture compound; and d) determining said analyte in a sample based onthe result of step c). The present invention further relates to a methodfor improving the specificity of an indirect immunoassay for determiningan analyte, comprising replacing at least 10% of a capture compound by anon-identical capture compound; wherein the capture compound replacedcompetes in binding to said analyte with the capture compoundintroduced. The present invention further relates to kits, devices, anduses related to the aforementioned methods.

BACKGROUND

Laboratory tests, in particular immunological tests, have becomeinvaluable tools in the diagnosis of disease. Immunoassays in particularhave provided the possibility to specifically detect single analytes orgroups of analytes in complex mixtures, e.g. body fluids, such as bloodor plasma. However, several causes of interference in immunoassays havebeen identified, e.g. cross-reactivity of an interfering substance witha capture compound, unspecific binding of detector compound to a solidphase, “bridge” binding of capture compound and detector compound byheterophile antibodies or, in particular, human anti-mouse antibodies(HAMA), to name a few (see, e.g. Park & Kricka (2013), Ch.5.3-Interferences in Immunoassay, in The Immunoassay Handbook (FourthEdition), edited by David Wild, Elsevier, Oxford: 403; Schiettecatte(2012), Interferences in Immunoassays, Advances in ImmunoassayTechnology, Dr. Norman H. L. Chiu (Ed.)).

In competitive immunoassays, analytes comprised in a sample compete witha labeled analyte (specifier) for binding to a capture compound, whichis frequently an antibody, or, in case the presence of antibodies toe.g. a pathogenic agent is to be tested in a serum sample, an antigen ofsaid pathogenic agent. In case the concentration of analyte in thesample is high, there is a strong competition, leading to a decreasedbinding of the specifier to the capture compound, causing signalreduction, which, depending on the test format, leads to a quantitativeor qualitative test result. It is a disadvantage of the immunoassaysknown in the art that antigens used as a capture compound may also bebound by interfering compounds from the sample. These interferingcompounds compete with the specifier for binding epitopes of the capturecompound. Depending on how severe the competition is, false test resultsare possible, causing a decrease of the specificity of the respectiveimmunoassay. This decrease in specificity can be especially pronouncedif complex capture compounds, potentially even consisting of severalsubunits with a large number of conformational epitopes, e.g. viralcapsids, are used. In such cases, the classical methods of interferenceelimination, e.g. addition of alternative targets for the interferingcompounds, may be inefficient.

In non-competitive immunoassays, the analyte is detected by contactingthe analyte to a compound specifically binding to the analyte and eithercarrying a label itself or being target of a second molecule carrying alabel. Thus, in non-competitive immunoassays, the amount of analyte isdetermined by determining the amount of complexes formed between theanalyte and a detector compound carrying a label. Accordingly, theanalogous specificity problems may be faced as described above.

Antigens being immunologically only slightly different (e.g. by aproduction in a different expression system, different amino acidsequence, difference in glycosylation, differences in purification andrefolding procedures, as well as differences in buffer and storageconditions) have an individual scope of interferences. Accordingly,samples leading to a false result in an immunoassay with antigen X1 mayprovide correct results in a test with antigen X2 and vice versa.Accordingly, exchanging an antigen for a different antigen frequentlydoes not lead to an improvement of specificity, but only to a change ofthe scope of samples leading to false results.

Problem to be Solved

It is therefore an objective of the present invention to provideimproved immunoassays avoiding the problems as described above.

SUMMARY OF THE INVENTION

These problems are solved by the methods, kits, devices, andcompositions with the features of the independent claims. Typicalembodiments, which might be realized in an isolated fashion or in anyarbitrary combination are listed in the dependent claims.

The present invention relates to a method for determining an analyte ina sample suspected to comprise said analyte, comprising a) contactingwith said sample at least a first and a second capture compound for saidanalyte, wherein said first and second capture compounds arenon-identical capture compounds, and wherein said capture compoundscompete in binding to said analyte; b) contacting said capture compoundscontacted with said sample with a specifier, wherein said specifiercompetes in binding to said capture compounds with said analyte; c)determining the amount of complexes comprising said specifier and acapture compound; and d) determining said analyte in a sample based onthe result of step c). The present invention further relates to a methodfor improving the specificity of an indirect immunoassay for determiningan analyte, comprising replacing at least 10% of a capture compound by anon-identical capture compound; wherein the capture compound replacedcompetes in binding to said analyte with the capture compoundintroduced. The present invention further relates to kits, devices, anduses related to the aforementioned methods.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for determining an analyte ina sample suspected to comprise

-   -   said analyte, comprising    -   a) contacting with said sample at least a first and a second        capture compound for said analyte, wherein said first and second        capture compounds are non-identical capture compounds, and        wherein said capture compounds compete in binding to said        analyte;    -   b) contacting said capture compounds contacted with said sample        with a specifier, wherein said specifier competes in binding to        said capture compounds with said analyte;    -   c) determining the amount of complexes comprising said specifier        and a capture compound; and    -   d) determining said analyte in a sample based on the result of        step c).

As used in the following, the terms “have”, “comprise” or “include” orany arbitrary grammatical variations thereof are used in a non-exclusiveway. Thus, these terms may both refer to a situation in which, besidesthe feature introduced by these terms, no further features are presentin the entity described in this context and to a situation in which oneor more further features are present. As an example, the expressions “Ahas B”, “A comprises B” and “A includes B” may both refer to a situationin which, besides B, no other element is present in A (i.e. a situationin which a solely and exclusively consists of B) and to a situation inwhich, besides B, one or more further elements are present in entity A,such as element C, elements C and D or even further elements.

Further, as used in the following, the terms “preferably”, “morepreferably”, “most preferably”, “particularly”, “more particularly”,“specifically”, “more specifically” or similar terms are used inconjunction with optional features, without restricting alternativepossibilities. Thus, features introduced by these terms are optionalfeatures and are not intended to restrict the scope of the claims in anyway. The invention may, as the skilled person will recognize, beperformed by using alternative features. Similarly, features introducedby “in an embodiment of the invention” or similar expressions areintended to be optional features, without any restriction regardingalternative embodiments of the invention, without any restrictionsregarding the scope of the invention and without any restrictionregarding the possibility of combining the features introduced in suchway with other optional or non-optional features of the invention. Theterm “about” in the context of specific values or ratios of the presentinvention refers to said value or ratio +/−30%, +/−20%, +/−10%, or, inan embodiment +/−5% of a given value or ratio.

The method of the present invention, in an embodiment, is an in vitromethod. Moreover, it may comprise steps in addition to the onesspecifically mentioned. In particular, step b) may comprise steps ofproviding a sample, or step c) may comprise addition of furthercompounds in order to facilitate binding and detection. Furthermore,some or all steps may be assisted by automated equipment.

As used herein, the term “determining” refers to determining at leastone immunological feature of an analyte to be determined by the methodof the present invention in the sample. Immunological features inaccordance with the present invention, in an embodiment, are structuralfeatures of the analyte facilitating detection of the analyte in asample by immunological means. In an embodiment, said immunologicalfeatures facilitate identification, in a further embodiment,quantification of the analyte by immunological means. Accordingly,typical immunological features are features facilitating differentiationof said analyte from other chemical compounds in a sample. In anembodiment, determining an analyte is establishing whether an analyte ispresent or absent in the sample at a concentration above the detectionlimit of the method. Methods of determining a detection limit are knownto the skilled person. In a further embodiment, determining isdetermining semi-quantitatively or quantitatively the amount orconcentration of an analyte in a sample. For quantitative determination,either the absolute or precise amount of the analyte will be determinedor the relative amount of the analyte will be determined. The relativeamount may be determined in a case were the precise amount of an analytecan or shall not be determined. In said case, it can be determinedwhether the amount in which the analyte is present is increased ordiminished with respect to a second sample comprising said analyte in asecond amount.

As will be understood by the skilled person, there will typically be norequirement to determine the first capture compound/analyte and secondcapture compound/analyte complexes separately. Thus, in an embodiment,the complexes of first capture compound and analyte and of the secondcapture compound and analyte are determined together, i.e.indiscriminative between said first and said second capture compound.Thus, in an embodiment, the total amount of analyte present in a complexwith either the first capture compound or the second capture compound isdetermined.

As will be further understood by the skilled person, the determinationof analyte/capture compound complexes will depend on the assay formatchosen. In an embodiment, the assay is a competitive immunoassay,typically a competitive, heterogeneous immunoassay, i.e. an immunoassay,wherein an analyte competes with a labeled derivative of said analytefor binding to a capture compound bound to a solid surface, and whereinthe amount of labeled derivative of said analyte bound to said capturecompound is determined. In an embodiment, the competitive assay is anassay for Anti-HBc, Anti-HAV (anti-Hepatitis A virus), Anti-HBe(anti-Hepatitis B e-antigen), Folate, Folate RBC (red blood cell),Anti-TSH-R, Vitamin D total, Vitamin B12, Tyroxin T4, FT 4 (freethyroxine), Tyroxin T3, FT 3 (free triiodothyronine), Testosteron,Progesterone, Digitoxin, Anti-TG, Anti-TPO (anti-Thyroid peroxidase),DGEA, or Estradiol. In a further embodiment, the immunoassay is adouble-antigen sandwich assay (“DAGS”) wherein a bivalent analyte, e.g.an antibody, is bound to a capture compound bound to a solid surface,and wherein the amount of analyte/capture compound complexes isdetermined by binding of a detector compound as specified herein belowto said analyte/capture compound complexes. In an embodiment, the DAGSassay is an assay for Anti-Toxoplasma IgG, Anti-rubella IgG, Anti-HBs1G, HCV (hepatitis C virus), e.g. HCVII, CMV (cytomegalovirus) IgG,Syphilis, HTLV (Human T-cell lymphotropic virus), or Chagas (Americantrypanosomiasis).

The term “biological molecule” is known to the skilled person and,typically, relates to a molecule produced by the metabolism of at leastone organism. Accordingly, the term “biological macromolecule” relatesto a polymer produced by an organism, in an embodiment, from monomericprecursors. A typical biological macromolecule is a polypeptide, DNA,RNA, or a polysaccharide.

The term “analyte”, as used herein, relates to a chemical molecule, inan embodiment, an organic molecule, binding to the capture compoundand/or detector compound of the present invention with sufficientaffinity to allow detection of an analyte/capture compound and/ordetector compound complex. In an embodiment, the dissociation constant(K_(d)) of the analyte/ligand complex is at most 10⁻⁷ mol/L, in afurther embodiment, at most 10⁻⁸ mol/1, in a further embodiment, at most10⁻⁹ mol/L. In an embodiment, the dissociation constant of the complexformed between the first capture compound of the invention and theanalyte and the dissociation constant of the complex formed between thesecond capture compound of the invention and the analyte are differentby not more than a factor of five; in an embodiment by no more than afactor of two; in a further embodiment by no more than a factor of 1.5.In an embodiment, the dissociation constant of the complex formedbetween the first capture compound and the analyte has a value of from70% to 130% of the dissociation constant of the complex formed betweenthe second capture compound and the analyte. In a further embodiment,the dissociation constant of the complex formed between the firstcapture compound and the analyte has a value of from 80% to 120% of thedissociation constant of the complex formed between the second capturecompound and the analyte. In a further embodiment, the dissociationconstant of the complex formed between the first capture compound andthe analyte has a value of from 90% to 110% of the dissociation constantof the complex formed between the second capture compound and theanalyte. In a further embodiment, the dissociation constant of thecomplex formed between the first capture compound and the analyte andthe dissociation constant of the complex formed between the secondcapture compound and the analyte are essentially equal or are equal. Inan embodiment, the analyte has a molecular mass of at least 100(corresponding to 100 atomic mass units, and to 100 Da; 1 Dacorresponding to 1.66×10⁻²⁷ kg), in a further embodiment, at least 250,in a further embodiment, at least 500, or, in a further embodiment, atleast 1000. In an embodiment, the analyte is a biological molecule, in afurther embodiment, the analyte is a biological macromolecule. In afurther embodiment, the analyte is a polypeptide.

In an embodiment, in case the analyte of the present invention is apolypeptide, the polypeptide is an antigen produced by an infectiousagent, e.g., a virus or bacterium, or an antibody, e.g., an antibodyproduced by a subject against an antigen produced by an infectiousagent. In an embodiment, the analyte is a polypeptide, in a furtherembodiment, an antibody against a viral antigen, in a furtherembodiment, against a viral polypeptide, or, in a further embodiment,against a viral capsid polypeptide. In an embodiment, the viral capsidpolypeptide is a Hepatitis virus capsid polypeptide, in an embodiment, aHepatitis B virus (HB) capsid polypeptide, or, in a further embodiment,a HB core (HBc) antigen. Accordingly, the analyte, in an embodiment, isan antibody against a Hepatitis virus capsid polypeptide, e.g., againsta Hepatitis B virus (HB) capsid polypeptide, typically, against a HBcore (HBc) antigen or a HBe antigen. In an embodiment, the first capturecompound is HBc antigen as shown in Genbank Acc No: Q17UT2 GI:123867942(SEQ ID NO: 1). In a further embodiment, the first capture compound isHBc antigen as shown in Genbank Acc No: Q17UT2 GI:123867942 (Hepatitis Bvirus subtype adw2 core antigen, SEQ ID NO: 1) and the second capturecompound is HBc antigen as shown in Genbank Acc No. P03147.1 GI:116947(Hepatitis B virus genotype D subtype adw core antigen, SEQ ID NO: 2).In a further embodiment the first and second capture compounds are HBcantigen sequences selected from HBc antigen sequences known in the artsuch as HBcAg protein sequences identified by their Genbank Acc. Nos:P03148.3 GI:166215076, P03149.1 GI:116945, NP_647607.1 GI:21326588, andAY123424.1 GI:22203511.

In an embodiment, to facilitate cloning, the N- and/or the C-terminalend of each HBV core antigen sequence may be shortened by deleting 1 to5 of the N- and/or C-terminal amino acids of said antigen sequence. In afurther embodiment, tags comprising 2 to 15 amino acids can be added toeither the N-terminal or the C-terminal end or to both ends of theantigen without interfering with the antigenic properties of the HBVcore antigen.

The term polypeptide, as used herein, in an embodiment, includesvariants and fragments of the specifically indicated polypeptides.Variants include polypeptides comprising amino acid sequences which areat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequences specifically indicated, e.g. shown in SEQ ID NO: 1 or 2. Thepercent identity values are, preferably, calculated over the entireamino acid sequence region. A series of programs based on a variety ofalgorithms is available to the skilled worker for comparing differentsequences. In this context, the algorithms of Needleman and Wunsch orSmith and Waterman give particularly reliable results. To carry out thesequence alignments, the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programsGap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970))and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))], which arepart of the GCG software packet [Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711 (1991)], are to be used. The sequenceidentity values recited above in percent (%) are to be determined,preferably, using the program GAP over the entire sequence region withthe following settings: Gap Weight: 50, Length Weight: 3, Average Match:10.000 and Average Mismatch: 0.000, which, unless otherwise specified,shall always be used as standard settings for sequence alignments.

A polypeptide comprising a fragment of any of the aforementionedpolypeptide sequences, in an embodiment, is also encompassed as apolypeptide of the present invention. The fragment be a polypeptidewhich still has the of being a capture compound or a detector compoundas specified above. Accordingly, the polypeptide may comprise or consistof the domains of the polypeptide of the present invention conferringthe said biological activity. A fragment as meant herein, preferably,comprises at least 50, at least 100, at least 250 or at least 500consecutive amino acid residues of any one of the aforementioned aminoacid sequences comprising at least 20, at least 30, at least 50, atleast 80, at least 100 or at least 150 consecutive amino acids of anyone of the aforementioned amino acid sequences.

The polypeptides of the present invention either essentially consist ofthe aforementioned amino acid sequences or comprise the aforementionedamino acid sequences. Thus, they may contain further polypeptidesequences as well. Specifically, the polypeptides of the presentinvention may be fusion proteins wherein one partner of the fusionprotein is a polypeptide as recited above. Such fusion proteins maycomprise as additional part other enzymes of the fatty acid or lipidbiosynthesis pathways, polypeptides for monitoring expression (e.g.,green, yellow, blue or red fluorescent proteins, alkaline phosphataseand the like) or so called “tags” which may serve as a detectable markeror as an auxiliary measure for purification purposes or for binding saidpolypeptides to a solid surface. Tags for the different purposes arewell known in the art and comprise FLAG-tags, 6-histidine-tags,MYC-tags, Biotin, and the like.

It is, however, also envisaged that the analyte is a low-molecularweight compound determinable by complex formation with a capturecompound, including, e.g. thyroxin (T4), triiodothyronine (T3), folicacid, folic acid binding protein, vitamin B₁₂, intrinsic factor, and thelike.

As used herein, the term “capture compound” relates to a chemicalmolecule binding, directly or indirectly, to the analyte of the presentinvention as specified herein above, and bound to a solid surface oradapted to be bound to a solid surface.

In an embodiment, the capture compound is an organic molecule, in afurther embodiment, a biological macromolecule as specified hereinabove, e.g., a polypeptide as specified herein above. In an embodiment,the capture compound binds indirectly to the analyte of the presentinvention with sufficient affinity to allow detection of the complexcomprising analyte and capture compound; i.e., in such case, the capturecompound is an indirect ligand. The term “indirect binding”, as usedherein, relates to a binding wherein the ligand does not directlycontact the analyte, but contacts a chemical molecule binding theanalyte, in an embodiment specifically binding the analyte, wherein, inan embodiment, said molecule binding the analyte is a molecule directlybinding the analyte, i.e. is a direct ligand. Accordingly, in anembodiment, the analyte, a chemical molecule binding the analyte, andthe indirect ligand form a complex with the properties as indicatedabove, in particular with the dissociation constants as indicated. Aswill be understood by the skilled person, the definition of the term“compete in binding” applies mutatis mutandis to the indirect ligands ofthe present invention, i.e. the indirect ligands, in an embodiment, havethe property of being unable to bind to said molecule binding theanalyte at the same time. In a further embodiment, the capture compounddirectly binds to the analyte of the present invention with sufficientaffinity to allow detection of the analyte/capture compound complex, asspecified herein above. Accordingly, in an embodiment, the capturecompound is a direct ligand.

According to the present invention, at least a first and a secondcapture compound are provided, wherein said first and second capturecompounds are non-identical capture compounds. As used herein, the term“non-identical” capture compounds relates to two or more species ofcapture compound molecules measurably different in at least one chemicaland/or physical property. Typically, said least one chemical and/orphysical property is not an indicator and is not a property related tobinding of said capture compound to a solid surface. Typical propertiesare amino acid sequence, glycosylation, three-dimensional folding and/orconformation, and length of polypeptide chain. However, also differencesin the concentration and/or identity of impurities in two preparationsof the same capture compound are contemplated by the present invention.Accordingly, in an embodiment, a second capture compound is derived orderivable from a first capture compound by at least one of (i)introducing at least one amino acid exchange into the amino acidsequence of said first capture compound, (ii) producing said secondcapture compound in a different cellular background as compared to saidfirst capture compound, (iii) removing or preventing glycosylation ofsaid second capture compound as compared to said first capture compound,(iv) purifying said second capture compound by different means ascompared to said first capture compound, (v) denaturing and/or refoldingsaid second capture compound under different conditions as compared tosaid first capture compound, and (vi) storing said second capturecompound under different conditions as compared to said first capturecompound. In an embodiment, said first and second capture compounds areantibodies recognizing essentially the same epitope and said secondcapture compound is a variant of an antibody being the first capturecompound. In a further embodiment, at least one of said first and secondcapture compounds is not an antibody. In an another embodiment, saidfirst and second capture compounds are not antibodies.

According to the present invention, at least a first and a secondcapture compound are provided, wherein said capture compounds compete inbinding to said analyte. As used herein, the term indicating that twocompounds “compete in binding” to an analyte relates to the property ofmolecules of said compounds of being unable to bind to essentially thesame binding site of said analyte at the same time. Thus, typically, incase two capture compounds compete in binding to an analyte and in casesaid analyte has one binding site for said capture compounds, at eachpoint in time, only one molecule of said capture compounds can be boundto said analyte. It will be understood by the skilled person that theabove applies mutatis mutandis in case the analyte has two or morebinding sites for the capture compounds. The skilled person knows how todetermine competition in binding. In an embodiment, competing in bindingto an analyte is binding to the same or essentially the samesubstructure of said analyte. In a further embodiment, in case theanalyte is a polypeptide, the epitope bound by a first capture compoundand the epitope bound by a second capture compound have at least 3 aminoacids, in an embodiment, 3 contiguous amino acids, of the analyte incommon. Typically, in such case, the epitope bound by a first capturecompound and the epitope bound by a second capture compound have atleast 4, 5, 6, or 7 amino acids of the analyte in common.

In an embodiment, the first and second capture compound do not competein binding to an interfering compound. The term “interfering compound”,as used herein, relates to a compound decreasing the specificity of animmunoassay. In an embodiment, the interfering compound is a compoundbinding to a capture compound of the present invention which is not theanalyte.

In an embodiment, the first and the second capture compound are used ata ratio of 1:5 to 5:1, typically 1:2 to 2:1, about 1:1, or 1:1. In afurther embodiment, three capture compounds are used at a ratio of about2:1:1, 1:2:1, or 1:1:2, about 1:1:1, or 1:1:1 Thus, in an embodiment,the first and the second capture compound and potentially presentfurther capture compounds, are present in the reaction mixture in aboutthe same amount, in a further embodiment, in the same amount.

Methods of binding biological molecules, typically polypeptides, tosolid surfaces are well known in the art and include, e.g. binding byhydrophobic interaction, biotinylation and binding via immobilizedstreptavidin, covalent binding, antibody-antigen interaction, and thelike, or a combination of these interactions, e.g. antibody-antigeninteraction between an antibody and a polypeptide of a pathogen, whereinsaid antibody is biotinylated and bound to a solid surface viaimmobilized streptavidin. Accordingly, the capture compound may,preferably, also be a capture complex. In an embodiment, the capturecompound is the compound of a capture complex directly binding to theanalyte. The skilled person knows how to bind a capture compound orcomplex to a solid surface, depending on the solid surface selected. Inan embodiment, the capture compound is a viral polypeptide, e.g., aviral capsid polypeptide. In an embodiment, said capture compound is aHepatitis virus capsid polypeptide, in an embodiment, a Hepatitis Bvirus (HB) capsid polypeptide, e.g., a HB core (HBc) antigen or a HBeantigen. It is, however, also envisaged that the capture compound is anantibody.

The term “antibody”, as used herein, includes monoclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired binding activity as specified elsewhere herein. Inan embodiment, an antibody is not an antibody comprised in an antiserum,typically not a polyclonal antibody or a polyclonal serum. Accordingly,in an embodiment, an antibody is an antibody comprised in a mixturewherein at least 80%, in an embodiment at least 90%, in a furtherembodiment, at least 95% of antibody molecules comprised in said mixtureare at least one capture compound or at least one detector compound ofthe present invention. In an embodiment, the antibody is a monoclonalantibody. In an embodiment, the antibody is a full-length antibody or anantibody fragment.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known and described generally in,for example, Abbas et al., Cellular and Mol. Immunology, 4th ed., W.B.Saunders, Co. (2000). An antibody may be part of a larger fusionmolecule, formed by covalent or non-covalent association of the antibodywith one or more other proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region. “Antibody fragments” comprise a portion of an intactantibody, in an embodiment, comprising the antigen-binding regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. “Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three hypervariableregions (HVRs) of each variable domain interact to define anantigen-binding site. Collectively, the six HVRs confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three HVRs specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site. The term “diabodies” refers to antibodyfragments with two antigen-binding sites, which fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) in the same polypeptide chain (VH-VL). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies may bebivalent or bispecific. Diabodies are described more fully in, forexample, EP 0 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9 (2003)129-134; and Hollinger et al., PNAS USA 90 (1993) 6444-6448. Triabodiesand tetrabodies are also described in Hudson et al., Nat. Med. 9 (2003)129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds an analyte, wherein the analyte-binding polypeptide sequence wasobtained by a process that includes the selection of a single analytebinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target-bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal-antibody preparation is directed against a single determinanton an antigen. In addition to their specificity, monoclonal-antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins.

As used herein, the term “solid surface” relates to any suitable solidsurface adapted for binding the capture compound of the presentinvention and adapted for being separated, e.g., by physical means, froma sample. In an embodiment, said solid surface is a surface of a bead,in an embodiment, a microbead, e.g. a magnetic or paramagneticmicrobead. In an embodiment, said surface is adapted to improve bindingof the capture compound, e.g. by attaching, covalently ornon-covalently, molecules binding a substructure of the capturecompound. Typical molecules binding a substructure of the capturecompound are, e.g. antibodies, streptavidin, complexed Nickel ions, andthe like. In a further embodiment, the solid surface binds said capturecompound by covalent or non-covalent bonds, e.g. by hydrophobicinteraction. Thus, in an embodiment, said solid surface is a surface ofa multi-cluster plate. In an embodiment, the surface of themulti-cluster plate is pretreated to increase affinity and/or capacityfor binding of a capture compound. Suitable pretreatments are known inthe art.

The term “sample”, as used herein, relates to a sample suspected tocomprise the analyte of the present invention. In an embodiment, thesample is or comprises a sample of a body fluid, a sample from a tissueor an organ, or a sample of wash/rinse fluid or a swab or smear obtainedfrom an outer or inner body surface. The sample, in an embodiment,comprises at least one analyte as specified elsewhere herein. Samples ofblood, plasma, serum, urine, saliva, or lacrimal fluid are encompassedby the method of the present invention. Samples can be obtained by useof brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsydevices, puncture of cavities with needles or lancets, or by surgicalinstrumentation. However, samples obtained by well known techniquesincluding, in an embodiment, scrapes, swabs or biopsies from theurogenital tract, perianal regions, anal canal, the oral cavity, theupper aerodigestive tract and the epidermis are also included as samplesof the present invention. Cell-free fluids may be obtained from the bodyfluids or the tissues or organs by lysing techniques such ashomogenization and/or by separating techniques such as filtration orcentrifugation. In an embodiment, samples are obtained from body fluidsknown to comprise HB virus polypeptides or/and antibodies against atleast one HB virus polypeptide, i.e., in an embodiment, blood, plasma,serum, saliva, or the like. It is to be understood that the sample maybe further processed in order to carry out the method of the presentinvention. Particularly, cells may be removed from the sample by methodsand means known in the art. Moreover, at least one analyte may beextracted and/or purified from the sample by methods and means known inthe art. Thus, the term sample also may relate to preparationscomprising or suspected to comprise at least one analyte, diluted,enriched, purified and/or extracted from a sample.

In an embodiment, the method of the present invention is a competitiveimmunoassay. Accordingly, the method further comprises admixing aspecifier to a sample. In an embodiment, the method of the presentinvention is a heterogeneous competitive immunoassay and comprisesindirectly determining the amount of complexes formed between an analyteand two non-identical capture compounds by determining the amount ofcomplexes formed between said specifier and said non-identical capturecompounds.

The term “contacting” as used in the context of the methods of thepresent invention is understood by the skilled person. In an embodiment,the term relates to bringing a compound of the present invention inphysical contact with a sample or with a further compound and thereby,e.g. allowing the sample and the compound to interact.

The term “specifier” is known to the skilled person and relates to acompound competing with an analyte for binding to the capture compounds,bonded to an indicator. Typically, the specifier is structurally similarto the analyte. Typically, the specifier comprises the substructure ofthe analyte bound by the capture compounds, bonded to an indicator. Inan embodiment, the specifier is a compound comprising the analyte and anindicator as structural elements, or the specifier consists of theanalyte covalently bonded to an indicator.

The term “indicator”, as used herein, is a compound adapted for makingthe presence of a molecule or complex comprising said indicatordetectable. Typically, the indicator has a detectable property,typically an optical or/and enzymatic property. It is, however, alsoenvisaged that said detectable property is the property of emittingradioactivity.

The term “optical property”, as used herein, relates to any propertywhich can be detected by an optical instrument. Specifically, theoptically determinable property may be or may comprise at least oneproperty selected from the group consisting of: a reflection property, atransmission property, an emission property, a scattering property, afluorescence property, a phosphorescence property, a diffractionproperty, and a polarization property. Further optical propertiesenvisaged by the present invention are color, fluorescence,luminescence, or refraction. In an embodiment, an optically determinableproperty as referred to herein refers to a property of a chemicalcompound which can be optically detected such as light absorption, lightemission, light remission, or properties associated therewith. It willbe understood that detecting an optically determinable property as usedherein encompasses the detection of the presence of a property which wasnot detectable before, the detection of the absence of a property whichhas been detected before, and the detection of quantitative changes of aproperty, i.e., the detection of the change of the signal strength whichcorrelates to the extent of the change of the at least one opticalproperty. It is understood that the term “optically determinableproperty”, in an embodiment, also relates to electrochemiluminescence,which is also known as electrogenerated chemiluminescence.

The term “enzymatic property”, as used herein, relates to a property ofan indicator of producing a detectable product from a substrate by meansof biological catalysis. Accordingly, an enzymatic property is typicallyconferred by the presence of a polypeptide having said enzymaticproperty in said indicator. Typically, the enzymatic property is atleast one enzymatic activity selected from the group consisting of:phosphatase activity (e.g. in alkaline phosphatase), peroxidase activity(e.g. in horseradish peroxidase), and glycosidase activity (e.g. inbeta-galactosidase). Typical substrates for enzymatic activities arewell-known in the art. Typically, said enzymatic activity produces aproduct having a determinable optical property as specified hereinabove, or/and said enzymatic activity produces a product beingdeterminable by an electrical instrument.

In an embodiment, the method of the present invention is adouble-antigen sandwich assay (“DAGS”). Accordingly, the method furthercomprises detecting complexes formed between said analyte and saidcapture compound by (i) contacting said complexes with at least onedetector compound and (ii) determining the amount of ternary complexescomprising said analyte, said capture compounds, and said detectorcompounds.

In the embodiment of a DAGS assay, a detector compound is used. The term“detector compound”, as used herein, relates to a chemical moleculebinding, directly or indirectly, to the analyte of the present inventionas specified herein above, and bonded to an indicator as specifiedelsewhere herein. In an embodiment, the detector compound is not boundto a solid surface and not adapted to be bound to a solid surface. Aswill be understood by the skilled person, the detector compound may alsobe an indirect detector compound, i.e. a detector compound notcontacting the analyte directly, as specified herein above for theligand of the invention. In an embodiment, the detector compound is adirect detector compound. Accordingly, in an embodiment, the definitionsprovided above for the capture compounds of the present invention, in asfar as they do not relate to binding of the compounds to a solidsurface, apply to the detector compounds of the present inventionmutatis mutandis.

As the skilled artisan will appreciate, the term “specific” is used toindicate that other compounds, typically biomolecules, present in asample do not significantly bind to a ligand (capture compound ordetector compound) of the present invention; as will be understood bythe skilled person, this does not exclude binding of chemical compoundsto regions of the capture compound or detector compound molecule notinvolved in interaction with the analyte, the specifier, or the tagserving as the functional moiety moderating the binding to a solidphase.

Moreover, the present invention relates to a method for improving thespecificity

(i) of an indirect immunoassay for determining an analyte, comprisingreplacing at least 10%, in an embodiment, at least 25%, in a furtherembodiment, at least 40%, in a further embodiment, at least 45%, in afurther embodiment, about 50% of a capture compound by a non-identicalcapture compound, wherein the capture compound replaced competes inbinding to said analyte with the capture compound introduced; or

(ii) of a double-antigen sandwich immunoassay for determining ananalyte,

comprising replacing at least 50%, in an embodiment, at least 75%, in afurther embodiment, at least 90%, in a further embodiment, about 100% ofa capture compound by a non-identical capture compound, wherein thecapture compound replaced competes in binding to said analyte with thecapture compound introduced and wherein said least one capture compoundand at least one detector compound are non-identical ligands of saidanalyte, or

comprising replacing at least 50%, in an embodiment, at least 75%, in afurther embodiment, at least 90%, in a further embodiment, about 100% ofa detector compound by a non-identical detector compound, wherein thedetector compound replaced competes in binding to said analyte with thedetector compound introduced. or of a detector compound and wherein saidleast one capture compound and at least one detector compound arenon-identical ligands of said analyte.

Typically, the fraction of capture compound or detector compoundreplaced will be selected such that a measurable effect of thereplacement can be expected theoretically. As will be understood by theskilled person, expectation of a measurable effect will depend on thenumber of non-identical ligands used for replacement. E.g. if in theassay after improvement three instead of one capture compounds are used,it is preferred that e.g. 66% of initial capture compound are replaced.As a general rule, it is envisaged that the fraction of a given capturecompound or detector compound is (100%/n)±50%, with n=(number ofnon-identical capture compounds or detector compounds used in an assay).In another embodiment, a fraction of (100%/n)±20% is used. As will beunderstood by the skilled person, the sum of fractions used will add upto 100%. Thus, in an embodiment, the fraction of capture compound ordetector compound replaced is of from 10% to 90%, in an embodiment, offrom 25% to 75%, in a further embodiment, of from 40% to 60%, in afurther embodiment, about 50%.

Furthermore, the present invention relates to a kit for detecting ananalyte in a sample, comprising

(i) at least two non-identical capture compounds for said analyte,wherein said capture compounds compete in binding to said analyte; or

(ii) at least two non-identical detector compounds for said analyte,wherein said detector compounds compete in binding to said analyte.

In an additional embodiment, the invention relates to a kit fordetecting an analyte in a sample, comprising

-   -   (i) at least two non-identical capture compounds for said        analyte, wherein said capture compounds compete in binding to        said analyte, and    -   (ii) a specifier, wherein said specifier competes in binding to        said capture compounds with said analyte.

The term “kit”, as used herein, refers to a collection of theaforementioned compounds, means or reagents of the present inventionwhich may or may not be packaged together. The components of the kit maybe comprised by separate vials (i.e. as a kit of separate parts) orprovided in a single vial.

Moreover, it is to be understood that the kit of the present inventionis to be used for practicing the methods referred to herein above. Itis, in an embodiment, envisaged that all components are provided in aready-to-use manner for practicing the methods referred to above.Further, the kit, in an embodiment, contains instructions for carryingout said methods. The instructions can be provided by a user's manual inpaper- or electronic form. For example, the manual may compriseinstructions for interpreting the results obtained when carrying out theaforementioned methods using the kit of the present invention. In anembodiment, the kit for detecting an analyte in a sample, comprising atleast two non-identical capture compounds of the invention furthercomprises at least two non-identical detector compounds, wherein saiddetector compounds compete in binding to said analyte, and wherein saidcapture compounds do not compete with said detector compounds in bindingto said analyte. In a further embodiment, the kit further comprises asolid support for immobilizing said capture compounds or forimmobilizing an analyte.

In an embodiment, of from 2 to 10 capture compounds for said analyte, inan embodiment of from 2 to 5 capture compounds for said analyte, in anembodiment of from 2 to 4 capture compounds, in an embodiment of from 2to 3 capture compounds for said analyte are comprised in said kit. In afurther embodiment, of from 2 to 10 detector compounds for said analyte,in an embodiment of from 2 to 5 detector compounds for said analyte, inan embodiment of from 2 to 4 detector compounds, in an embodiment offrom 2 to 3 detector compounds for said analyte are comprised in saidkit.

Also, the present invention relates to a device for determining ananalyte in a sample, comprising

(i) at least two non-identical capture compounds for said analyte,wherein said capture compounds compete in binding to said analyte; or

(ii) at least two non-identical detector compounds for said analyte,wherein said detector compounds compete in binding to said analyte, andoptionally means for determining an optical or/and enzymatic property ofan indicator comprised in said detector compounds.

The term “device”, as used herein, relates to a system of meanscomprising at least the aforementioned means operatively linked to eachother as to allow the determination. Typical means for determining theamounts of an analyte, and means for carrying out the determination aredisclosed above in connection with the methods of the invention. How tolink the means in an operating manner will depend on the type of meansincluded into the device. In an embodiment, the means are comprised by asingle device. Said device may accordingly include (i) an analyzing unitfor the measurement of the amount of the analyte in an applied sampleand a (ii) computer unit for processing the resulting data for theevaluation. Typical means for detection are disclosed in connection withembodiments relating to the method of the invention above. In such acase, the means are operatively linked in that the user of the systembrings together the result of the determination of the optically or/andelectrochemically determinable property of an indicator and the of theanalyte due to the instructions and interpretations given in a manual,or said instructions and interpretations are comprised in an executableprogram code comprised in the device, such that, as a result ofdetermination, an amount or concentration of analyte in the sampleapplied is output to the user. The person skilled in the art willrealize how to link the means without further ado. Typical devices arethose which can be applied without the particular knowledge of aspecialized technician, e.g., test stripes or electronic devices whichmerely require loading with a sample. The results may be given as outputof raw data which need interpretation by a technician. In an embodiment,the output of the device is, however, processed, i.e. evaluated, rawdata, the interpretation of which does not require a technician. Furthertypical devices comprise the analyzing units/devices (e.g., biosensors,arrays, solid supports coupled to ligands specifically recognizing thepeptide, Plasmon surface resonance devices, NMR spectrometers,mass-spectrometers etc.) or evaluation units/devices referred to abovein accordance with the method of the invention.

In an embodiment, of from 2 to 10 capture compounds for said analyte, inan embodiment of from 2 to 5 capture compounds for said analyte, in anembodiment of from 2 to 4 capture compounds, in an embodiment of from 2to 3 capture compounds for said analyte are comprised in said device. Ina further embodiment, of from 2 to 10 detector compounds for saidanalyte, in an embodiment of from 2 to 5 detector compounds for saidanalyte, in an embodiment of from 2 to 4 detector compounds, in anembodiment of from 2 to 3 detector compounds for said analyte arecomprised in said device.

Moreover, the present invention relates to a composition comprising (i)at least two non-identical capture compounds for an analyte indicativeof disease, wherein said capture compounds compete in binding to saidanalyte; or (ii) at least two non-identical detector compounds for ananalyte indicative of disease, wherein said detector compounds competein binding to said analyte; for use in diagnosis.

The term “composition”, as used herein, refers to any compositionformulated in solid, liquid or gaseous form. Said composition comprisesthe compounds of the invention, optionally together with suitableauxiliary compounds such as diluents or carriers or further ingredients.Suitable diluents and/or carriers depend on the purpose for which thecomposition is to be used and on the other ingredients. The personskilled in the art can determine such suitable diluents and/or carrierswithout further ado. In an embodiment, the composition for use indiagnosis comprising at least two non-identical capture compounds of theinvention further comprises at least two non-identical detectorcompounds, wherein said detector compounds compete in binding to saidanalyte, and wherein said capture compounds do not compete with saiddetector compounds in binding to said analyte.

The term “diagnosis” as used herein refers to an assessment of theprobability according to which a subject is suffering or will sufferfrom a disease or condition. As will be understood by those skilled inthe art, such an assessment is usually not intended to be correct for100% of the subjects to be diagnosed. The term, however, requires that astatistically significant portion of subjects can be correctly diagnosedto suffer from the disease or condition. Whether a portion isstatistically significant can be determined without further ado by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test etc., Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, theprobability envisaged by the present invention allows that the diagnosiswill be correct for at least 60%, at least 70%, at least 80%, or atleast 90% of the subjects of a given cohort or population. Typicalconditions to be diagnosed are, e.g. fertility status or pregnancy.Typical diseases to be diagnosed are, e.g. prevalent or previousinfection with a pathogen, e.g. a virus, hyper- or hypothyroidism,vitamin deficiency and the like.

Moreover, the present invention relates to the use of a compositioncomprising (i) a first and a second capture compound, wherein said firstand second capture compounds are non-identical capture compounds, andwherein said capture compounds compete in binding to said analyte, or(ii) a first and a second detector compound, wherein said first andsecond detector compounds are non-identical detector compounds, andwherein said detector compounds compete in binding to said analyte; fordetermining an analyte in a sample.

Summarizing the findings of the present invention, the followingembodiments are disclosed:

Embodiment 1. A method for determining an analyte in a sample suspectedto comprise said analyte, comprising

-   -   a) contacting with said sample at least a first and a second        capture compound for said analyte, wherein said first and second        capture compounds are non-identical capture compounds, and        wherein said capture compounds compete in binding to said        analyte;    -   b) contacting said capture compounds contacted with said sample

(i) with a detector compound, wherein said detector compound competes inbinding to said analyte with said capture compounds; or

(ii) with a specifier, wherein said specifier competes in binding tosaid capture compounds with said analyte;

-   -   c) determining the amount of complexes comprising

(i) said detector compound and a capture compound, or

(ii) said specifier and a capture compound; and

-   -   d) determining said analyte in a sample based on the result of        step c).

Embodiment 2. The method of embodiment 1, wherein competing in bindingto a molecule is binding to essentially the same or to the samesubstructure of said molecule, in an embodiment, wherein competing inbinding to said analyte is binding to essentially the same or to thesame substructure of said analyte, in an embodiment, wherein competingin binding to said capture compounds is binding to essentially the sameor to the same substructure of said at least one, in an embodiment, allcapture compounds.

Embodiment 3. The method of embodiment 1 or 2, wherein saidnon-identical capture compounds are biological molecules, in anembodiment, biological macromolecules, in a further embodiment,polypeptides.

Embodiment 4. The method of any one of embodiments 1 to 3, wherein saidtwo non-identical capture compounds are variants of a viral polypeptide,in an embodiment, of a viral capsid polypeptide.

Embodiment 5. The method of any one of embodiments 1 to 4, wherein saidtwo non-identical capture compounds are variants of a Hepatitis viruscapsid polypeptide, in an embodiment, of a Hepatitis B virus (HB) capsidpolypeptide, in a further embodiment, of a HB core (HBc) antigen.

Embodiment 6. The method of any one of embodiments 1 to 2, wherein saidsecond capture compound is a variant of an antibody being the firstcapture compound; or wherein said first and second capture compounds areantibodies recognizing essentially the same epitope.

Embodiment 7. The method of any one of embodiments 1 to 6, wherein saidsecond capture compound is derived or derivable from said first capturecompound by at least one of:

-   -   (i) introducing at least one amino acid exchange into the amino        acid sequence of said first capture compound,    -   (ii) producing said second capture compound in a different        cellular background as compared to said first capture compound,    -   (iii) removing or preventing glycosylation of said second        capture compound as compared to said first capture compound,    -   (iv) purifying said second capture compound by different means        and/or by a different method as compared to said first capture        compound,    -   (v) denaturing and/or refolding said second capture compound        under different conditions as compared to said first capture        compound, and    -   (vi) storing said second capture compound under different        conditions as compared to said first capture compound.

Embodiment 8. The method of any one of embodiments 1 to 7, wherein saidanalyte is a biological macromolecule.

Embodiment 9. The method of any one of embodiments 1 to 8, wherein saidanalyte is a polypeptide.

Embodiment 10. The method of any one of embodiments 1 to 9, wherein saidanalyte is an antibody against a viral antigen, in an embodiment,against a viral polypeptide, in a further embodiment, against a viralcapsid polypeptide.

Embodiment 11. The method of any one of embodiments 1 to 10, whereinsaid analyte is an antibody against a Hepatitis virus capsidpolypeptide, in an embodiment, against a Hepatitis B virus (HB) capsidpolypeptide, in a further embodiment, against a HB core (HBc) antigen.

Embodiment 12. The method of any one of embodiments 9 to 11, wherein theepitopes bound by said capture compounds have at least 3 amino acids incommon.

Embodiment 13. The method of any one of embodiments 1 to 12, whereinsaid determining an analyte is qualitatively or quantitativelydetermining the amount of said analyte.

Embodiment 14. The method of any one of embodiments 1 to 13, whereinstep a) comprises contacting with said sample of from 2 to 10 capturecompounds for said analyte, in an embodiment of from 2 to 5 capturecompounds for said analyte, in an embodiment of from 2 to 4 capturecompounds, in an embodiment of from 2 to 3 capture compounds for saidanalyte.

Embodiment 15. The method of any one of embodiments 1 to 14, whereinsaid specifier comprises the substructure of the analyte bound by thecapture compounds, bonded to an indicator.

Embodiment 16. The method of any one of embodiments 1 to 15, whereinsaid specifier is a compound comprising the analyte and an indicator.

Embodiment 17. The method of any one of embodiments 1 to 16 wherein saidspecifier consists of the analyte covalently bonded to an indicator.

Embodiment 18: The method of any one of embodiments 1 to 17, whereinsaid capture compounds are not comprised in a polyclonal antiserum, inan embodiment, are not polyclonal antibodies.

Embodiment 19. The method of any one of embodiments 1 to 18, whereinsaid first and second capture compounds do not compete in binding to aninterfering compound.

Embodiment 20. The method of any one of embodiments 1 to 19, comprisingthe steps of:

a) contacting with said sample at least a first and a second capturecompound for said analyte, wherein said first and second capturecompounds are non-identical capture compounds, and wherein said capturecompounds compete in binding to said analyte;

b) contacting said capture compounds contacted with said sample with aspecifier, wherein said specifier competes in binding to said capturecompounds with said analyte;

c) determining the amount of complexes comprising said specifier and acapture compound; and

d) determining said analyte in a sample based on the result of step c).

Embodiment 21. A method for determining an analyte in a sample suspectedto comprise said analyte, comprising

-   -   a) contacting said sample to at least a first and a second        detector compound for said analyte, wherein said first and        second detector compounds are non-identical detector compounds,        and wherein said detector compounds compete in binding to said        analyte;    -   b) binding constituents of said sample including at least said        analyte to a solid surface,    -   c) determining the amount of complexes comprising a detector        compound bound to said solid surface; and    -   d) determining said analyte in a sample based on the result of        step c).

Embodiment 22. The method of embodiment 22, wherein said analytecomprises an antibody, in an embodiment, is an antibody.

Embodiment 23. The method of any one of embodiments 21 to 22, whereinsaid detector compound further comprises an indicator.

Embodiment 24. The method of any one of embodiments 15 to 17 or 23,wherein said indicator is a compound having a detectable property, in anembodiment, an optical or/and enzymatic property.

Embodiment 25. The method of any one of embodiments 20 to 24, whereinsaid first and second detector compound do not compete in binding to aninterfering compound.

Embodiment 26. The method of any one of embodiments 20 to 25, whereinstep a) is performed after step b).

Embodiment 27. A method for improving the specificity

(a) of (i) an indirect immunoassay for determining an analyte or (ii) adouble-antigen sandwich immunoassay for determining an analyte,comprising:

replacing at least 10%, in an embodiment, at least 25%, in a furtherembodiment, at least 40%, in a further embodiment, at least 45%, in afurther embodiment, about 50% of a capture compound by a non-identicalcapture compound, wherein the capture compound replaced competes inbinding to said analyte with the capture compound introduced; or

(b) of a double-antigen sandwich immunoassay for determining an analyte,comprising:

replacing at least 25%, in an embodiment, at least 40%, in a furtherembodiment, at least 45%, in a further embodiment, about 50% of adetector compound by a non-identical detector compound, wherein thedetector compound replaced competes in binding to said analyte with thedetector compound introduced and wherein said least one capture compoundand at least one detector compound are non-identical capture compoundsof said analyte.

Embodiment 28. The method of any one of embodiments 1 to 27, wherein thechemical structure in said capture compounds and/or said detectorcompounds binding to said analyte is identical in all of saidnon-identical capture compounds and/or in all of said non-identicaldetector compounds.

Embodiment 29. A kit for detecting an analyte in a sample, comprising

(i) at least two non-identical capture compounds for said analyte, in anembodiment of from 2 to 10 capture compounds for said analyte, in anembodiment of from 2 to 5 capture compounds for said analyte, in anembodiment of from 2 to 4 capture compounds, in an embodiment of from 2to 3 capture compounds, for said analyte, wherein said capture compoundscompete in binding to said analyte; or

(ii) at least two non-identical detector compounds for said analyte, inan embodiment of from 2 to 10 detector compounds for said analyte, in anembodiment of from 2 to 5 detector compounds for said analyte, in anembodiment of from 2 to 4 detector compounds, in an embodiment of from 2to 3 detector compounds, for said analyte, wherein said detectorcompounds compete in binding to said analyte.

Embodiment 30. The kit of embodiment 29, further comprising a solidsupport for immobilizing said capture compounds or constituents of saidsample comprising at least said analyte.

Embodiment 31. A device for determining an analyte in a sample,comprising

(i) at least two non-identical capture compounds for said analyte, in anembodiment of from 2 to 10 capture compounds for said analyte, in anembodiment of from 2 to 5 capture compounds for said analyte, in anembodiment of from 2 to 4 capture compounds, in an embodiment of from 2to 3 capture compounds, for said analyte, wherein said capture compoundscompete in binding to said analyte; or

(ii) at least two non-identical detector compounds for said analyte, inan embodiment of from 2 to 10 detector compounds for said analyte, in anembodiment of from 2 to 5 detector compounds for said analyte, in anembodiment of from 2 to 4 detector compounds, in an embodiment of from 2to 3 detector compounds, for said analyte, wherein said detectorcompounds compete in binding to said analyte and optionally means fordetermining an optical or/and enzymatic property of an indicatorcomprised in said detector compounds.

Embodiment 32. A composition comprising

(i) at least two non-identical capture compounds for an analyteindicative of disease, wherein said capture compounds compete in bindingto said analyte, or

(ii) at least two non-identical detector compounds for an analyteindicative of disease, wherein said detector compounds compete inbinding to said analyte;

for use in diagnosis of disease.

Embodiment 33. Use of

(i) at least two non-identical capture compounds for an analyte, whereinsaid capture compounds compete in binding to said analyte; and/or

(ii) at least two non-identical detector compounds for an analyte,wherein said detector compounds compete in binding to said analyte;

for the manufacture of a diagnostic composition or a diagnostic device.

Embodiment 34. The composition for use of embodiment 32 or use ofembodiment 33, wherein said disease is viral hepatitis.

Embodiment 35. Use of a composition comprising (i) a first and a secondcapture compound, wherein said first and second capture compounds arenon-identical capture compounds, and wherein said capture compoundscompete in binding to said analyte; or (ii) a first and a seconddetector compound, wherein said first and second detector compounds arenon-identical detector compounds, and wherein said detector compoundscompete in binding to said analyte; for determining an analyte in asample.

SHORT DESCRIPTION OF THE FIGURES

Further optional features and embodiments of the invention will bedisclosed in more detail in the subsequent description of embodiments,in an embodiment, in conjunction with the dependent claims. Therein, therespective optional features may be realized in an isolated fashion aswell as in any arbitrary feasible combination, as the skilled personwill realize. The scope of the invention is not restricted by thedisclosed embodiments. The embodiments are schematically depicted in theFigures. Therein, identical reference numbers in these Figures refer toidentical or functionally comparable elements.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H schematically indicate the principle of the presentinvention as applied to a competitive assay; a: analyte, s: specifier,c1: capture compound 1, c2: capture compound 2, i1: interfering agent 1,i2: interfering agent 2. FIG. 1A) In the absence of the analyte, thespecifier binds to capture compound 1. FIG. 1B) In the presence of theanalyte, the analyte binds to capture compound 1 and prevents thespecifier from binding to capture compound 1. FIG. 1C) In the presenceof interfering agent 1, having affinity to capture compound 1 (but notto capture compound 2), interfering agent 1 binds to capture compound 1,thus preventing the specifier from binding. FIG. 1D) In the presence ofinterfering agent 2, having affinity to capture compound 2 (but not tocapture compound 1), interfering agent 2 does not bind to capturecompound 1, thus permitting the specifier to bind to capture compound 1.Conversely, FIG. 1E) interfering agent 1, having affinity to capturecompound 1 (but not to capture compound 2), does not bind to capturecompound 2, thus permitting the specifier to bind to capture compound 2;and FIG. 1F) interfering agent 2 does bind to capture compound 2, thuspreventing the specifier from binding. (FIGS. 1G) and 1H)) in casecapture compound 1 and 2 are used e.g. at a 1:1 ratio, the false signalreduction induced by interfering agent 1 or interfering agent 2 will bereduced to approximately 50%.

FIGS. 2A & 2B schematically show the test principle of a competitivetest for anti-Hepatitis core (HBc) antigen antibodies. HBc: Hepatitis Bcore antigen; α-HBc: anti-HBc antigen antibody; the non-conjugated α-HBcantibody depicted with a double circumference is an antibody potentiallypresent in a sample from a patient, the conjugated antibodies depictedwith a single circumference are α-HBc antibodies added during the assay;conjugation is either with Ru: Ruthenium-complex, or bio: biotin; strp:streptavidin-coating of a solid support. FIG. 2A) in the absence of theanalyte (a-HBc antibody), both, Ru-conjugated and bio-conjugated α-HBcantibodies can bind to HBc added to the assay mixture. Via thebio-strp-interaction, the complex is bound to a solid support and afterwashing, a signal generated from the Ru-conjugation can be measured.FIG. 2B) in the presence of the analyte (a-HBc antibody), the α-HBc-bioantibody and Ru-conjugated antibody are prevented from binding to HBcadded to the assay mixture, thus preventing binding of a complex to thesolid support. Accordingly, after washing, no signal can be generatedfrom the Ru-complex, since this will be washed off. In FIGS. 2A & 2B,HBc as well as the α-HBc-bio conjugate are part of the capture compoundcomplex.

FIGS. 3A-3H schematically indicates the principle of the presentinvention as applied to a double-antigen sandwich (DAGS) format. a:analyte, d: detector compound, c1: capture compound 1, c2: capturecompound 2, i1: interfering agent 1, i2: interfering agent 2. FIG. 3A)in the absence of analyte, no analyte is bound to capture compound 1 (or2) fixed on a solid support and, thus, the detector compound will bewashed away in washing steps. FIG. 3B) in the presence of the analyte,e.g. an antibody, the analyte binds to capture compound 1 and to thedetector compound, thus mediating binding of the detector compound tothe solid surface. FIG. 3C) in the presence of interfering agent 1,which binds to capture compound 1 and to the detector compound, but notto capture compound 2, the detector compound becomes fixed to the solidsupport via the interaction with interfering agent 1. FIG. 3D) in thepresence of interfering agent 2, which binds to capture compound 2 andto the detector compound, but not to capture compound 1, the detectorcompound is not fixed to the solid support via interfering agent 1.Conversely, FIG. 3E) in the presence of interfering agent 1, thedetector compound is not fixed to the solid support via the interactionwith interfering agent 2; and FIG. 3F) in the presence of interferingagent 2, the detector compound will be fixed to the solid support viacapture compound 2. (FIGS. 3G) and 3H)) in case capture compound 1 and 2are used e.g. at a 1:1 ratio, the false signal increase induced byinterfering agent 1 or interfering agent 2 will be reduced toapproximately 50%. A scheme could mutatis mutandis be drawn for the useof two non-identical detector compounds.

EXAMPLES Example 1

Cloning and Purification of Recombinant Hepatitis B Core Antigen

The synthetic gene encoding the Hepatitis B core antigen (HBcAg) waspurchased from Eurofins MWG Operon (Ebersberg, Germany). On the basis ofthe pET24a expression plasmid of Novagen (Madison, Wis., USA) thefollowing cloning steps were performed. The vector was digested withBamH1 and Xho1 and a cassette comprising the HBcAg was inserted. Theinsert of the resulting plasmid was sequenced and found to encode thedesired protein. The amino acid sequence of the resulting protein isshown in the sequence protocol of the present invention. The recombinantHBcAg did not contain a C-terminal hexahistidine tag.

The recombinant HBcAg was purified according to the following protocol.E. coli BL21 (DE3) cells harboring the expression plasmid were grown inLB medium plus kanamycin (30 μg/ml) to an OD600 of 1, and cytosolicoverexpression was induced by adding isopropyl-β-D-thiogalactosid (IPTG)to a final concentration of 1 mM at a growth temperature of 37° C. 4hours after induction, cells were harvested by centrifugation (20 min at5000×g), frozen and stored at −20° C. For cell lysis, the frozen pelletwas resuspended in 25 mM sodium phosphate pH 8.5, 6 mM MgCl2, 10 Um′Benzonase®, 1 tablet Complete® and 1 tablet Complete® EDTA-free per 50ml of buffer (protease inhibitor cocktail) and the resulting suspensionwas lysed by high pressure homogenization. The crude lysate wascentrifuged and the HBcAg in the supernatant was precipitated withammonium sulfate (35% w/v).

After additional centrifugation the precipitate was resuspended anddialyzed against a phosphate buffer followed by a heating step (70° C.for 30 minutes). After centrifugation the clear supernatant was appliedonto a Toyopearl DEAE 650-11 column (from Tosoh Bioscience)pre-equilibrated in 25 mM potassium phosphate pH 7. The protein was theneluted by applying a gradient up to a potassium chloride concentrationof 500 mM. Finally, the protein was subjected to size exclusionchromatography (S400) and the protein-containing fractions were pooled.

Example 2

Immunoassay for Detecting Anti-HBc Antibodies in a Competitive TestFormat

Serum samples from a panel of healthy blood donors were analyzed in acompetitive Anti-HBc ECLIA (Electrochemiluminescence Immunoassay) forthe presence of anti-HBc antibodies in automated Cobas e® analyzers(Roche Diagnostics GmbH). Cobas e® and Elecsys® are registeredtrademarks of the Roche group. The samples were tested negative forAnti-HBc with at least one CE-marked Anti-HBc assay that is differentfrom the Elecsys® Anti-HBc assay and which is commercially available.

In the assay, serum is reacted with HBc. After addition of biotinylatedantibodies and ruthenium complex(Tris(2,2′-bipyridyl)ruthenium(II)-complex; (Ru(bpy)32+)-labeledantibodies, both specific for HBcAg, together with streptavidin-coatedmicroparticles, the still-free binding sites on the HBc-antigens becomeoccupied. The entire complex becomes bound to the solid phase viainteraction of biotin and streptavidin. After removal of unboundsubstances, a voltage is applied to the electrode and induceschemiluminescent emission at 620 nm after excitation at a platinumelectrode which is measured by a photomultiplier.

High measured values indicate binding of biotinylated antibodies andruthenium complex-labeled antibodies added and, thus, absence ofanti-HBc antibodies in the sample. In the presence of anti-HBcantibodies in the sample these antibodies compete with both types ofassay specific antibodies for binding to the antigen HBcAG leading toreduced light emission at 620 nm after excitation at a platinumelectrode. The signal output is in arbitrary light units.

Accordingly, samples having a measured value of higher than the cut-offindex 1.0 are considered non-reactive, whereas samples having a measuredvalue lower than or equal to the cut-off index 1.0 are consideredreactive.

Based on the competitive Elecsys® Anti-HBc assay format three differentHBcAg settings were tested. “HBcAg X” refers to an antigen comprisingSEQ ID NO:2; “HBcAg Y” refers to an antigen comprising SEQ ID NO:1. Onlythe antigen setting was modified, all other reagent and conditionsremained unchanged. The results of true positive (infected) samples wasnot affected by applying a combination of HBcAg X and Y as first andsecond capture compound (data not shown). Table 1 relates only todiscrepant (=false) positive results of the sample panel. The threedifferent settings were as follows:

A) HBcAg X is used as target in the competitive Anti-HBc assay.

B) HBcAg Y (slightly different from HBcAg X) is used as target in thecompetitive Anti-HBc assay.

C) A combination of HBcAg X and HBcAg Y are used together as target inthe competitive anti-HBc assay

Table 1 shows the results of a competitive Anti-HBc-ECLIA(Electrochemiluminescence Immunoassay). Commercially available sera(Bavarian Red Cross) negative for antibodies against HBc antigen wereused as samples. Table 1 lists only those results with discrepantfindings. Different HBcAG showed individual patterns of discrepantpositive Anti-HBc results. When two different preparations ofHBc-antigen (HBcAG) were used, 11 samples were tested (false-) positive(reactive) when HBcAG X was used, 7 samples tested (false-) positiveonly when the second HBcAG Y was used, and 2 samples tested(false)-positive with both HBcAG X and with HBcAG Y. In contrast, when a1:1 mixture of both antigens, i.e. HBcAG X and HBcAG Y was used, only 5of these 18 samples tested positive, decreasing the number offalse-positive tests by more than 3-fold. In detail, the number ofsamples tested false-positive using HBcAGX only, was reduced from 11 to3 and the number of samples tested false-positive using HBcAGY only, wasreduced from 9 to 5 when said 1:1 mixture of both antigens (HBcAG X andHBcAG Y) was used. As expected, the two samples tested false-positivewith both antigens (Samples No. 2942 and 3657), also testedfalse-positive with the mixture.

As a consequence, the specificity of this competitive immunoassay wasincreased when at least two slightly different HBc antigens were appliedas a mixture.

By applying the principle of using two slightly different first andsecond capture compounds for an analyte in a method based on acompetitive test format the specificity can be considerably increased,i.e. false positive results can be avoided to a considerable extent.

TABLE 1 Results of a competitive Anti-HBc ECLIA(Electrochemiluminescence Immunoassay), COI: Cut-off index HBcAG MixInterpretation HBcAG X HBcAG Y X + Y reactive COI ≤ 1.0 COI ≤ 1.0 COI ≤1.0 non-reactive COI > 1.0 COI > 1.0 COI > 1.0 Sample No. COI COI COIAnti-HBcAG X 1984 0.234* 2.19 1.19 interference 2942 0.463* 0.462*0.396* 3180 0.582* 1.37 0.976* 3633 0.639* 2.17 1.29 3470 0.788* 2.261.38 3784 0.820* 2.17 1.47 2426 0.865* 1.53 1.06 3657 0.878* 0.984*0.858* 3976 0.891* 2.34 1.41 2423 0.927* 2.16 1.52 2905 0.990* 2.22 1.51Anti-HBcAG Y 2858 1.66 0.185* 0.941* interference 3813 1.59 0.311* 1.071344 1.39 0.428* 0.910* 2038 2.05 0.473* 1.21 2898 2.03 0.754* 1.38 15291.83 0.800* 1.34 2263 1.56 0.840* 1.20 Anti-HBc 2243 1.84 2.26 2.09negative 2269 1.94 2.26 2.04 2155 2.02 2.26 2.13 1311 1.98 2.26 2.081065 2.01 2.26 2.26 *Discrepant 11 9 5 positive samples [n]

1-13. (canceled)
 14. A kit for detecting an analyte in a sample,comprising (i) at least two non-identical capture compounds for saidanalyte, or (ii) at least two non-identical detector compounds for saidanalyte, wherein said at least two non-identical capture compounds ordetector compounds are non-identical polypeptides differing in at leastone property selected from the group consisting of amino acid sequence,glycosylation, three-dimensional folding and/or conformation, and lengthof polypeptide chain, and wherein said at least two non-identicalcapture compounds or detector compounds compete in binding to saidanalyte.
 15. The kit of claim 14, wherein said at least twonon-identical capture compounds or said at least two non-identicaldetector compounds comprise the same analyte binding domain.
 16. The kitof claim 14, wherein said kit comprises at least two non-identicalcapture compounds.
 17. The kit of claim 16, wherein said at least twonon-identical capture compounds have amino acid sequences sharing atleast 70% sequence identity.
 18. The kit of claim 16, wherein said atleast two non-identical capture compounds are bound to a solid surfaceor are adapted to be bound to a solid surface.
 19. The kit of claim 16,wherein said kit further comprises at least one antibody forming capturecomplexes with said at least two capture complexes.
 20. The kit of claim19, wherein said at least one antibody is bound to a solid surface or isadapted to be bound to a solid surface.
 21. The kit of claim 16, whereinsaid at least two non-identical capture compounds are direct ligands ofsaid analyte.
 22. The kit of claim 14, wherein said kit comprises offrom 2 to 10 capture compounds for said analyte, in an embodiment offrom 2 to 5 capture compounds for said analyte, in an embodiment of from2 to 4 capture compounds, in an embodiment of from 2 to 3 capturecompounds, for said analyte.
 23. The kit of claim 16, wherein said kitfurther comprises a specifier.
 24. The kit of claim 23, wherein saidspecifier comprises the substructure of the analyte bound by the atleast two non-identical capture compounds, bonded to an indicator. 25.The kit of claim 23, wherein the specifier consists of the analytecovalently bonded to an indicator.
 26. The kit of claim 14, wherein saidkit comprises at least two non-identical detector compounds, saiddetector compounds each comprising said analyte binding domain and anindicator.
 27. The kit of claim 26, wherein said at least twonon-identical detector compounds have amino acid sequences sharing atleast 70% sequence identity.
 28. The kit of claim 26, wherein said atleast two non-identical detector compounds comprise the same indicator.29. The kit of claim 26, wherein said indicator has a detectableproperty selected from the group consisting of an optical property, anenzymatic property, and a property of emitting radioactivity.
 30. Thekit of claim 26, wherein said at least two non-identical detectorcompounds are direct ligands of said analyte.
 31. The kit of claim 14,wherein said kit comprises of from 2 to 10 detector compounds for saidanalyte, in an embodiment of from 2 to 5 detector compounds for saidanalyte, in an embodiment of from 2 to 4 detector compounds, in anembodiment of from 2 to 3 detector compounds, for said analyte.
 32. Thekit of claim 14, wherein said at least two non-identical capturecompounds or said at least two non-identical detection compounds do notcompete in binding to an interfering compound.
 33. The kit of claim 14,wherein a first capture compound or a first detector compound comprisesan amino acid sequence at least 70% identical to SEQ ID NO:1, and/orwherein a second capture compound or a second detector compoundcomprises an amino acid sequence at least 70% identical to SEQ ID NO:2.